The general aim of this proposal is to elucidate the role of glycosylphosphatidylinositol (GPI), glycosphingolipids (GSLs) and cholesterol in the processes that lead to the polarized structure of epithelial cells and neurons. Our laboratory has shown that proteins anchored via GPI are apically targeted in various kidney and intestinal epithelial cells (MDCK, LLC-PK, Caco-2), and that this is due to specific sorting mechanisms operating in the TGN. This proposal aims to investigate the nature of the mechanisms that regulate the incorporation of GPI- proteins and GSLs into apical carrier vesicles and to identify sorter molecules in the TGN that participate in this process. For this purpose, we will utilize cell lines characterized by our laboratory that sort differentially GPI-proteins and GSLs. VIP21/caveolin, a protein present in TGN-derived vesicles and in plasma membrane caveolae, appears as a major player in the clustering of GPI-anchored proteins with GSLs and cholesterol, and in the poorly understood interactions of these clusters with the apical sorting machinery. The role of VIP21 /caveolin in TGN sorting will be studied by transfection of the protein into a rat thyroid cell line (FRT) which does not express it, fails to sort GPI-anchored proteins apically and fails to cluster GPI-proteins with GSLs and cholesterol. Deep-etch electron microscopy will be used to look for the characteristic striated coat of caveolae in the plasma membrane and Golgi complex in wild type FRT and in FRT cells expressing caveolin. A video microscopy assay will be used to search for endogenous cross-linking factors that may be necessary for the recruitment of GPI-proteins into caveolae or their equivalent post-TGN vesicles. Cross-linking experiments with bifunctional reagents and mechanical isolation of plasma membrane and Golgi caveolae will be attempted to identify components of the apical sorting machinery that interact with GSL patches and GPI-anchored proteins. We will also compare the polarity of GPI-proteins in epithelial cells and neurons and develop procedures to characterize the surface distribution of free GPI and CSLs in epithelial cells. It is expected that these studies will clarify important aspects of the biology of epithelial cells and neurons.